WO1999013109A1 - Procede d'analyse de sequençage d'adn - Google Patents

Procede d'analyse de sequençage d'adn Download PDF

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Publication number
WO1999013109A1
WO1999013109A1 PCT/US1998/018808 US9818808W WO9913109A1 WO 1999013109 A1 WO1999013109 A1 WO 1999013109A1 US 9818808 W US9818808 W US 9818808W WO 9913109 A1 WO9913109 A1 WO 9913109A1
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Prior art keywords
nucleotides
products
substrate
fluorescence
cleaved
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PCT/US1998/018808
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English (en)
Inventor
John J. Macklin
Jay K. Trautman
Timothy D. Harris
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Seq, Ltd.
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Publication date
Application filed by Seq, Ltd. filed Critical Seq, Ltd.
Priority to AU94762/98A priority Critical patent/AU9476298A/en
Publication of WO1999013109A1 publication Critical patent/WO1999013109A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention is a method for identifying one or more of the different types of nucleotides in the resulting sequence of cleaved nucleotides.
  • the cleaved nucleotides are affixed to a substrate so that each nucleotide is located at a different position on the substrate in the same order in which it was cleaved.
  • the affixed nucleotides are then contacted with reagents and may be illuminated with electromagnetic radiation so as to selectively convert one or more of the different types of nucleotides in place to products that have enhanced fluorescence.
  • the conversion product for each type of nucleotide advantageously has a fluorescence characteristic (e.g., wavelength of maximum absorption or emission, or fluorescence lifetime) that is different from the fluorescence characteristic of the other conversion products.
  • the conversion products are illuminated with electromagnetic radiation at a wavelength that is suitable for causing the conversion product to fluoresce.
  • the fluorescence of each conversion product is detected as a function of the position of the conversion product on the substrate. Where the fluorescence of each conversion product is distinctive, detection of all the conversion products can be done simultaneously. Where the fluorescence of two or more conversion products is not distinctive, each such product is formed and detected at a different time.
  • the result of the foregoing process is a map that indicates the position on the substrate of the different conversion products that were caused to fluoresce. Since the position of the conversion products corresponds to the position of the nucleotides that were affixed to the substrate and since the nucleotides were deposited on the substrate in the sequence in which they were cleaved, the map identifies the position in the oligonucleotide of those nucleotides that were converted to conversion products whose fluorescence was detected.
  • nucleotides are converted to conversion products having distinctive fluorescence.
  • the invention may also be practiced where only one, two or three nucleotides are converted to conversion products having distinctive fluorescence. While information relating to the location of only one, two or three nucleotides in an oligonucleotide sequence may not be complete, it is still useful.
  • information relating to the location of only one, two or three nucleotides in an oligonucleotide sequence may not be complete, it is still useful.
  • information as to the position of the other two bases can be obtained by sequencing the complementary strand.
  • Fig. 1 depicts a system useful in cleaving nucleotides one-at-a-time from an oligonucleotide
  • Fig. 2 (a) is an enlarged side view of a portion of the system of Fig. 1 depicting the cleavage of nucleotides one- at-a-time from an oligonucleotide;
  • Fig. 2 (b) is a top view of the same portion of the system depicted by Fig. 2 (a) ;
  • Fig. 2 (c) is a top view of a substrate to which the cleaved nucleotides have been affixed;
  • Fig. 3 depicts a system for detecting the fluorescence from conversion products formed in accordance with the invention
  • Figs. 4A-4E depict illustrative maps of the position of the nucleotides on the substrate;
  • Fig. 5 depicts an alternative system for forming a sequence of nucleotides on a substrate;
  • Fig. 6 is a schematic representation of several chemical compounds ; and Fig. 7(a), 7(b) and 7(c) show fluorescent spectra for two modified nucleotides illuminated at two different wavelengths .
  • Fig. 1 depicts an optical trap useful in practicing a preferred embodiment of the invention.
  • Fig. 1 is reproduced from U.S. Patent 5,079,169 which is incorporated herein by reference and the elements of Fig. 1 bear the same reference numerals .
  • Fig. 1 depicts a conventional optical trap 10 of the type which is used in practicing the invention.
  • the trap comprises a modified fluorescence microscope 12 including a chamber 14 containing a liquid cell where particle manipulation takes place.
  • the chamber is mounted on a conventional microscope stage 16 which can be moved in two orthogonal directions in the plane perpendicular to the axis of the microscope, as well as along the optical axis.
  • the optical trap is formed by a laser beam from a laser 22 which is focused on chamber 14 by a highly convergent objective lens 48.
  • the laser is an argon ion laser, a diode laser or a NdYAG laser.
  • Lens 48 typically has a numerical aperture greater than 0.8 and preferably about 1.2 or greater.
  • lens 48 is a liquid immersion type and an oil drop between lens 48 and the cover of chamber 14 approximately matches the refractive indices of the lens and the cover so as to minimize light losses at the surfaces.
  • the position of the optical trap in chamber 14 may be moved by moving platform 24 in the X or Y directions. Alternatively, stage 16 may be moved with respect to the optical trap.
  • the apparatus of Fig. 1 further comprises an image intensified video camera 60 or other electro-optical imaging device, a fluorescence light source 62 such as an argon laser or a mercury lamp and a visible light source 66. Further details concerning this optical trap are set forth in the '169 patent. A similar such trap is illustrated in Fig. 1 of U.S. Patent 4,893,886 which is likewise incorporated herein by reference.
  • an optical trap may be used to draw an oligonucleotide through a thin film on a substrate.
  • an oligonucleotide such as a strand of DNA may be attached at one end to a small bead using known methods and the bead may be secured in the trap.
  • the bead and oligonucleotide may then be drawn through the thin film by moving the substrate relative to the trap, by moving the trap, or by moving both.
  • Fig. 2 (a) and 2 (b) show a thin film 80 on a substrate 82, an optical trap 84 in which is secured a bead 86 and an oligonucleotide 88 attached to the bead.
  • nucleotides 92 By attaching a processive exonuclease 90 to the free end of the oligonucleotide and providing conditions in the thin film such that the exonuclease is activated, individual nucleotides 92 will be cleaved from the oligonucleotide in the same order in which they are found in the oligonucleotide. Absolute position of the nucleotides is referenced to unique position markers 94 attached to the surface.
  • each of the cleaved nucleotides is deposited in the film at a different position but in the same order in which it is found in the oligonucleotide.
  • a microchannel is defined in the surface of the substrate and the oligonucleotide is drawn through a thin film in this channel. The channel minimizes lateral diffusion of the nucleotides and therefore aids in defining their position on the substrate.
  • each of the cleaved nucleotides is affixed to the substrate in the order in which it was deposited in the film.
  • this is effected by electrostatic attraction between the substrate which advantageously is made of alumina and the negative phosphate group of the nucleotides. It may be further promoted by an applied electric field or other means discussed below.
  • Fig. 2 (c) depicts substrate 82 to which a sequence of nucleotides has been affixed.
  • one or more of the bases and preferably all four are converted to conversion products having a fluorescence that is substantially enhanced over that of the native nucleotide.
  • this conversion is preferably effected by contacting the substrate with solutions containing suitable reagents and, when the conversion product is formed from a photochemical reaction, illuminating the substrate with uv radiation. This can be done using a separate solution for each of the four nucleotides followed by a washing step or it may be possible to combine in one solution suitable reagents for converting more than one of the nucleotides to the desired conversion products.
  • Fig. 3 is essentially a microscope with a CCD readout.
  • this apparatus comprises a substrate 82 to which the conversion products are affixed, a quartz coverslip 115, a high numerical aperture objective lens 120, a dichroic mirror 125, first and second CCD cameras 130, 135 and a CPU 140 for controlling the microscope and processing the data received by the CCD cameras.
  • the substrate is covered with a detection solution 112 which typically is an aqueous buffer solution free of dissolved oxygen and containing a triplet quencher.
  • the substrate is mounted on a microscope stage which is movable in at least one dimension and, in particular, is movable along the same pathway that the oligonucleotide was drawn when the nucleotide were cleaved from the oligonucleotide and affixed to the substrate.
  • the movement of the microscope stage is controlled by CPU 140.
  • the substrate is illuminated from below by a source of electromagnetic radiation, preferably a laser (not shown) , having a wavelength suitable for stimulating fluorescence in the conversion products affixed to the substrate.
  • a suitable laser is a mode-locked Ti: sapphire laser, frequency-tripled into the ultraviolet, that produces tunable wavelengths from about 260 nanometers (nm) to 295 nm with 100 to 200 milliwatts (mW) of power.
  • Alternative sources may be an Ar- ion laser which is capable of producing 3 mW at 275 nm and 10 W at 300 nm and a Hg lamp which produces about 5 mW at 240- 260 nm.
  • Electromagnetic radiation is evanescently coupled to the conversion products so that the intensity of the radiation decreases exponentially with distance into the detection solution. As a result, any absorbing molecules in the detection solution are minimally excited, thereby minimizing unwanted background fluorescence. This also allows molecules to interact with the bound conversion products and not be depleted by the ultraviolet light.
  • the microscope is set to image a portion of the substrate at a time, about a 250-micron field of view for a lOOx objective. A slightly smaller fraction of the field is illuminated by the uv light.
  • the resulting spatially isolated fluorescence from the photoproducts is imaged onto a CCD camera, with suitable optical filters placed in the detection path to preferentially pass only the wavelengths of interest.
  • the uv intensity is about 50 W/cm 2 .
  • the CCD camera and the uv light is shuttered on for about one second then shuttered off. This produces about 50 photons per molecule detected as spatially resolved spots on the CCD image, or frame.
  • the substrate is then moved in the direction that the original DNA strand was dragged, by an amount slightly smaller than the field of view, so that the uv excitation light slightly overlaps the previous field and the present field of view; and the lumination process is repeated.
  • the apparatus of Fig. 3 may be used in a variety of ways to detect the conversion products on substrate 82. Ideally, all four conversion products will have sufficiently different fluorescence emission characteristics that they can be detected simultaneously. As an aid to this type of operation, both CCD cameras may be used for simultaneous detection with different wavelength filters 132, 137 inserted in their optical paths. This approach has the advantage of avoiding registration problems that otherwise might occur when combining two or more successive images of the conversion products on substrate 82.
  • the invention may also be practiced by monitoring the fluorescence of only one, two or three of the conversion products at one time and then combining the images that are generated.
  • Monitoring the fluorescence of only one conversion product at a time has the advantage that the illuminating radiation may be tuned to a wavelength that has maximum absorption by only one of the conversion products, thereby enhancing the fluorescence from that conversion product and improving its detectability by the CCD camera.
  • each conversion product is separately illuminated, four images are produced which must then be combined to produce a single map depicting the sequence of the nucleotides as affixed to the substrate.
  • four illustrative images that would be detected are shown in Figs. 4A, 4B, 4C and 4D for the four bases A,C,G and T and the combined image is illustrated in Fig. 4E.
  • spectroscopic characteristics of the conversion products can be additionally used for discrimination in the arrangement shown in Fig. 3. For example, it may be desirable to time-resolve the fluorescence from the nucleotides, since the fluorescent lifetime may be a unique characteristic of the modified nucleotides.
  • a sample containing the conversion products is illuminated with electromagnetic radiation that causes the products to fluoresce, where the illumination is from a pulsed light source, for example a mode-locked laser, where the pulse duration is shorter than the shortest fluorescent decay of the modified nucleotides, while the time between pulses is much longer than the longest fluorescent decay.
  • a fluorescence image of the sample is formed on a time-resolved fluorescence detector from a unique spatial location in the field of view.
  • the decay time for each channel (or spatial location) is determined, and a fluorescence-lifetime image is produced.
  • This image can then be used to determine the spatial location and identity of a conversion product, based on the lifetime recorded at each pixel.
  • This method of discrimination may be used in addition to spectral filtering to provide a higher confidence level in the unique identification of a conversion product.
  • the detection arrangement described above could be modified to incorporate confocal illumination and detection, where the uv illumination beam is focused to a small spot on the sample, and all the fluorescence emission from the sample at that spot, after suitable optical filtering, is sent to a single channel detector, for example, a MCP detector or an avalanche-photodiode.
  • a single channel detector for example, a MCP detector or an avalanche-photodiode.
  • the fluorescence decay at each spot on the sample is analyzed by software to determine the presence and identity of the conversion product, based on the optically-filtered fluorescence decay time.
  • the sample is rastered so that all spots on the sample are probed.
  • This detection scheme can be extended to incorporate a slit confocal geometry, which is intermediate between a full-field geometry and a single-point confocal geometry.
  • oligonucleotide may be suspended in a flowing stream of solution with an active exonuclease attached to its free end. See, for example, U.S. patent application Serial No. 08/376,761, which is incorporated herein by reference. In this situation, cleaved nucleotides become entrained in the flowing stream and may be deposited in sequence on a substrate by apparatus such as that shown in Fig. 5.
  • this apparatus establishes on the substrate a sequence of nucleotides in the order in which they were cleaved from the oligonucleotide.
  • the flowing stream is deposited by a nozzle 300 in a thin continuous liquid film 305 or as discrete droplets on a transparent support 310.
  • the liquid film or droplets are then solidified on the solid support by cooling the support and/or polymerization and/or drying.
  • the film is transported by movement of support 310 through a detection station 320 where it is irradiated by a radiation source 325; and the resulting fluorescence is detected by detection system 330 and identified by computer 335.
  • the specific structure of detection station 320 may be similar to the apparatus of Fig. 3.
  • the support can take the form of any surface geometry which can be moved with respect to the output nozzle 300 so as to allow for the deposit of the nucleotide containing liquid stream in such a manner that the position of deposit of each nucleotide is both unique and known (Merrill et al., 1979, J. Histochem. Cytoche . 27:280-283, which is incorporated herein by reference) .
  • Uniqueness requires that individual nucleotides are deposited on the surface with sufficient distance between each nucleotide and any other nucleotide so as to be isolated in an optically-resolvable volume element during the subsequent step of nucleotide detection and identification infra .
  • the sequential position of deposit of each nucleotide must be known with sufficient accuracy so as to be able to position the volume element containing each nucleotide with respect to the excitation volume of the detection system infra .
  • the detection of nucleotides in a continuous film 305 or in discrete droplets on a support 310 is similar to the detection of nucleotides in the apparatus of Fig. 3. As indicated in Fig. 5, film 305 (or droplets) is moved through a beam of radiation from a radiation source 325 and fluorescence is detected by a detector system 330. However, in this case the cross-sectional dimension of film 305 (or of the droplets) is almost certain to be much greater than that in the apparatus of Fig. 3.
  • Fig. 5 provides additional possibilities for performance of the step of converting the nucleotides to conversion products with enhanced fluorescence.
  • one or more reagents for effecting the conversion can be introduced into the flowing stream upstream of the nozzle. If the activity of the exonuclease is not inhibited by such reagents, the reagents can be introduced into the stream upstream of the exonuclease. Otherwise, they can be introduced downstream of the point where the nucleotides are cleaved from the oligonucleotide.
  • nucleotides While it is preferred that all of the nucleotides first be converted to conversion products before any of the conversion products are illuminated for detection, the invention may also be practiced by performing one or more conversions, illuminating the substrate to detect one or more of the conversion products and then converting one or more other nucleotides to conversion products. Binding and Immobilization of Nucleotides to Surfaces
  • the immobilization of monophosphate nucleotides at a liquid/solid interface following their sequential cleavage from a strand of DNA represents a useful method for their subsequent detection at the single molecule level.
  • metal oxides such as aluminum oxide (alumina) preferentially binds nucleotides but not nucleosides or bases 13 where binding results from the electrostatic attraction of the negative phosphate group of a nucleotide with the tri-valent positively charged aluminum ions on the alumina surface. Because the binding occurs through the phosphate group all of the monophosphate nucleotides (dAMP, dCMP, dGMP, and TMP) bind with nearly equal binding affinity. Examples of possible coordination of phosphate on alumina are discussed in Refs.
  • alumina-bound nucleotides can remain bound to the surface for hours, with a 1/e off-rate of about 1/(7 hrs) at room temperature. This could be further reduced using a less polar solvent to overcoat the nucleotides.
  • changing the solution buffer does not appear to displace the nucleotides from the surface, unless a very acidic solution or concentrated phosphate-buffer solution is used.
  • Photophysical measurements show that the fluorescence properties of surface-bound nucleotides are essentially unchanged from those found in solution. There is no indication of undesirable processes such as charge- or energy-transfer from the nucleotide to the surface.
  • the substrate containing the spatially-ordered surface- immobilized nucleotides can be processed to further decrease the off-rate and diffusion of the bound nucleotides.
  • a solution containing 'blocking' molecules that bind to the alumina can be spread over the surface.
  • a blocking molecule could be riboside-monophosphate (a nucleotide without a base) that binds up the remaining available sites on the alumina surface, without displacing the nucleotides.
  • the blocking molecules act to (1) prevent binding of reagent molecules in subsequent processing steps, and (2) further reduce the diffusion of nucleotides along the surface (by analogy to the restricted movement of cars in a filled parking lot) .
  • a method to enhance the fluorescence properties of native nucleotides preferably does not involve the nucleotide phosphate group, which is used to bind the nucleotide to a substrate. This can be accomplished by a chemical or photochemical reaction between the nucleotide and a non- fluorescent reagent that results in a fluorescent product. This product is a uv-excited, near-uv emissive fluorophore with high quantum yield. This method does not require aggressive removal of the reagent after the reaction; the non-fluorescent reagent will not obscure the visibility or detectability of the fluorescent product.
  • Figure 6 shows the molecular structure of the four bases of DNA, along with four fluorescent products following chemical modification of the bases.
  • the spectroscopic properties of the fluorescent products are listed in Table I.
  • a particularly simple chemical synthesis is the reaction of chloroacetal with dAMP to form the highly fluorescent (quantum yield of 56%) l,N 6 -ethenoadenosine monophosphate, or etheno-dAMP.
  • This reaction in solution proceeds to complete conversion with no side reaction in excess chloroacetal.
  • We have verified that the conversion reagents do not generate appreciable background when applied to a crystalline alumina binding surface.
  • the methods for chemical and photochemical conversion can be ordered to optimize the fluorescence detection process while minimizing the decomposition of the product molecules.
  • the photochemical conversion of dGMP to the dGMP-photoproduct will not produce significant photodecomposition of dAMP. This may be accomplished by choice of the wavelength of the radiation used for the photochemistry.
  • the dGMP nucleotide may be photoconverted to the dGMP-photoproduct using, for example, 280 nm light, which lies outside the absorption band of dAMP and has little deleterious effect on dAMP nucleotides. A map of the spatial location of the dGMP-products can be obtained.
  • a chemical modification step is employed to convert dAMP to etheno-dAMP, where chemistry used may produce little modification of the dGMP-photoproduct.
  • the photochemical and chemical conversion may be first performed, followed by the detection and discrimination of the conversion products in a subsequent step.
  • Fig. 7(a) shows the emission and excitation spectra for a dGMP photoproduct made by photochemical reaction, and for etheno-dAMP, the chemically- modified analog of dAMP. It can be seen that the two emission spectra are distinguishable, so that using excitation at, say, 300 nm, the dGMP-photoproduct can be distinguished from the dAMP-product by simple optical filters in front of the detector (s).
  • a sample could first be excited at 265 nm, where the etheno-dAMP absorbance is maximum and the dGMP- photoproduct absorbance is minimum.
  • the resulting fluorescence (Fig. 7(b)) would arise almost exclusively from the etheno-dAMP, and an optical filter which passes 385-485 nm would further aid in the discrimination.
  • the sample would then be illuminated with, say, 300 nm (Fig. 7(c)), and an optical filter transmitting 330 nm - 385 nm switched into the path of the detected fluorescence, so that the detected signal would be due almost exclusively to the dGMP- photoproduct.
  • a time-resolved fluorescent image could also be formed, since the fluorescence decay time for etheno-dAMP (20 nsec) , and for dGMP-photoproduct (6.8 nsec) are quite different, and a software decision be made that at a particular spatial location, either dAMP was detected or dGMP was detected or no nucleotide was detected.

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Abstract

La présente invention concerne des nucléotides coupés, fixés à un substrat de sorte que chaque nucléotide est placé dans une position différente sur le substrat selon l'ordre dans lequel ils ont été coupés. Les nucléotides fixés sont ensuite mis en contact avec des réactifs qui transforment sélectivement un ou plusieurs types différents de nucléotides présents en produits qui améliorent la fluorescence. Lorsque plus d'un type de nucléotide est ainsi transformé, le produit de transformation pour chaque type de nucléotide présente avantageusement une caractéristique de fluorescence (par exemple, longueur d'onde d'absorption ou d'émission maximum, ou durée de vie de fluorescence) différente de la caractéristique de fluorescence des autres produits de transformation. Les produits de transformation sont éclairés par un rayonnement électromagnétique d'une longueur d'ondes permettant de provoquer une émission de fluorescence par le produit de transformation. La fluorescence de chaque produit de transformation est détectée sous forme d'une fonction de la position du produit de transformation sur le substrat. Lorsque la fluorescence de chaque produit de transformation est caractéristique, la détection de tous les produits de transformation peut être effectuée simultanément. Lorsque la fluorescence de deux ou plusieurs produits de transformation n'est pas caractéristique, chaque produit doit être détecté à un moment différent. Le procédé précédent produit une carte qui indique la position des différents produits de transformation qui ont provoqués la fluorescence. Du fait que la position des produits de transformation correspond à la position des nucléotides fixés au substrat et du fait que les nucléotides déposés sur le substrat dans la séquence dans laquelle ils ont été coupés, la carte identifie la position dans l'oligonucléotide des nucléotides transformés en produits de transformation dont la fluorescence a été détectée.
PCT/US1998/018808 1997-09-11 1998-09-10 Procede d'analyse de sequençage d'adn WO1999013109A1 (fr)

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AU94762/98A AU9476298A (en) 1997-09-11 1998-09-10 Method for dna sequencing analysis

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US60/058,642 1997-09-11

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WO2002002795A2 (fr) * 2000-06-30 2002-01-10 Gnothis Holding Sa Procede de sequençage multiplex
EP1330650A2 (fr) * 2000-10-12 2003-07-30 Amnis Corporation Procede et appareil de lecture de billes marquees par rapporteurs
WO2003080861A1 (fr) * 2002-03-22 2003-10-02 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Sequençage de molecule unique au moyen de nucleotides marques par des phosphates
US7138121B2 (en) 2003-01-23 2006-11-21 Spangler Brenda D Biosensors utilizing dendrimer-immobilized ligands and there use thereof
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US7645596B2 (en) 1998-05-01 2010-01-12 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
US7935481B1 (en) 1999-07-26 2011-05-03 Osmetech Technology Inc. Sequence determination of nucleic acids using electronic detection
US7981604B2 (en) 2004-02-19 2011-07-19 California Institute Of Technology Methods and kits for analyzing polynucleotide sequences
US9012144B2 (en) 2003-11-12 2015-04-21 Fluidigm Corporation Short cycle methods for sequencing polynucleotides
US9096898B2 (en) 1998-05-01 2015-08-04 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

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US5525464A (en) * 1987-04-01 1996-06-11 Hyseq, Inc. Method of sequencing by hybridization of oligonucleotide probes

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US5525464A (en) * 1987-04-01 1996-06-11 Hyseq, Inc. Method of sequencing by hybridization of oligonucleotide probes
WO1995021266A1 (fr) * 1994-02-01 1995-08-10 The Regents Of The University Of California Sondes marquees avec des colorants couples par transfert d'energie

Cited By (24)

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US10208341B2 (en) 1998-05-01 2019-02-19 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9096898B2 (en) 1998-05-01 2015-08-04 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9957561B2 (en) 1998-05-01 2018-05-01 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9212393B2 (en) 1998-05-01 2015-12-15 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9725764B2 (en) 1998-05-01 2017-08-08 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9540689B2 (en) 1998-05-01 2017-01-10 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9458500B2 (en) 1998-05-01 2016-10-04 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7645596B2 (en) 1998-05-01 2010-01-12 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US10214774B2 (en) 1998-05-01 2019-02-26 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7501245B2 (en) 1999-06-28 2009-03-10 Helicos Biosciences Corp. Methods and apparatuses for analyzing polynucleotide sequences
US7935481B1 (en) 1999-07-26 2011-05-03 Osmetech Technology Inc. Sequence determination of nucleic acids using electronic detection
US6596483B1 (en) 1999-11-12 2003-07-22 Motorola, Inc. System and method for detecting molecules using an active pixel sensor
WO2001035080A1 (fr) * 1999-11-12 2001-05-17 Motorola, Inc. Systeme et procede de detection de molecules a l'aide d'un capteur a pixels actif
WO2002002795A3 (fr) * 2000-06-30 2002-07-18 Gnothis Holding Sa Procede de sequençage multiplex
WO2002002795A2 (fr) * 2000-06-30 2002-01-10 Gnothis Holding Sa Procede de sequençage multiplex
EP1330650A2 (fr) * 2000-10-12 2003-07-30 Amnis Corporation Procede et appareil de lecture de billes marquees par rapporteurs
EP1330650B1 (fr) * 2000-10-12 2011-12-28 Amnis Corporation Procede de lecture de billes marquees par rapporteurs
WO2003080861A1 (fr) * 2002-03-22 2003-10-02 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Sequençage de molecule unique au moyen de nucleotides marques par des phosphates
US7138121B2 (en) 2003-01-23 2006-11-21 Spangler Brenda D Biosensors utilizing dendrimer-immobilized ligands and there use thereof
US9657344B2 (en) 2003-11-12 2017-05-23 Fluidigm Corporation Short cycle methods for sequencing polynucleotides
US9012144B2 (en) 2003-11-12 2015-04-21 Fluidigm Corporation Short cycle methods for sequencing polynucleotides
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US9868978B2 (en) 2005-08-26 2018-01-16 Fluidigm Corporation Single molecule sequencing of captured nucleic acids
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids

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